Growth Factor Independent (Gfi)-genes, Gfi-1 and Gfi-lb, are sites of retroviral insertions in murine lymphomagenesis and encode zinc-finger transcription factors. Homologs of Gfi-proteins in C. elegans and Drosophila regulate cell fate decisions in the nervous system. Within the mammalian hematopoietic system Gfi-1 and Gfi-1 b are expressed in a complementary pattern with Gfi-1 highly expressed in granulocytic and lymphoid cells, and Gfi-lb within erythroid and megakaryocytic lineages. Gene targeting experiments from this laboratory demonstrated that Gfi-1 is essential for granulocyte maturation and for down-regulation of macrophage properties. Gfi-lb is required for both erythroid and megakaryocytic differentiation, resembling in some aspects of GATA-1 deficiency. The overall goal of this proposal is to use a combined genetic and biochemical approach to link the Gfi-factors to specific regulatory networks and pathways within hematopoietic stem cells (HSCs) and their progeny. Aim 1 focuses on genetic dissection of functions of the Gfi-proteins in hematopoiesis. This will involved a detailed structure-function analysis of Gfi-1 and Gf-lb using rescue of gene targeted cells or mice, assessment of interchangeability of these factors through targeted gene knock-ins in mice, evaluation of the role of a novel isoform of Gfi-1 b (designated Gfi-lbL), and determination of the effects of Gfi-1, Gfi-lb, or combined deficiency on HSC competitive repopulation. Aim 2 focuses on biochemical approaches to the role of Gfi-proteins in hematopoietic cells. A method for in vivo biotinylation of expressed proteins will be used to facilitate efficient purification of proteins associating with Gfi-1 in myeloid cells and with Gfi-lb in erythroid and megakaryocytic cells. Gfi-associated proteins will be identified by mass-spectrometry microsequencing. Candidate interacting proteins will be further validated by coimmunoprecipitation assays and permit the Gfi-proteins to be placed within known regulatory pathways. In a second approach direct target genes of the Gfi-proteins will be sought by chromatin-purification from cells expressing biotin-tagged Gfi-proteins. Candidate target DNA regions will be identified by sequencing and will be validated by conventional chromatin-immunoprecipitation assays. The significance of target genes will be further assessed using gene targeted cells and mice. This research program should elucidate how Gfi-proteins control hematopoietic differentiation and how their activation contributes to oncogenesis.